Comparison of synthetic mycolactone A/B and its remote diastereomer.

<p>L929 fibroblasts and SW10 Schwann cells were cultured and treated with the same concentration of mycolactone A/B or mycolactone A/B remote diastereomer. Trypan blue staining and the TUNEL assay were performed. Synthetic mycolactone A/B (A, B) and its remote diastereomer (C, D) exerted identical cytotoxicity in both fibroblasts and Schwann cells at the same concentration.</p

Effect of mycolactone on cellular morphology.

<p>SW10 mouse Schwann cells and L929 mouse fibroblast cells were cultured for 24 hrs. Synthetic mycolactone A/B diluted to a final concentration of 3 ng/ml, 30 ng/ml, or 300 ng/ml was added to a cell culture and incubated for 12, 24, 48 and 72 hrs. Photomicrographs were taken by a phase-contrast microscope. Cells treated with 3 ng/ml of mycolactone showed no floating cells at 24, 48, or 72 hrs (A, B). Fibroblasts showed no changes until 48 hrs, but partial detachment began at 72 hrs with 30 ng/ml of mycolactone A/B (C). Schwann cells showed round shrinkage and floating at 24 hrs with 30 ng/ml of mycolactone A/B. Some of the cells remained adherent at 48 hrs, but all cells were detached at 72 hrs (D). Bar = 100 μm.</p

Detection of apoptosis by fluorescence microscopy.

<p>Fibroblasts and Schwann cells were cultured in chamber slides for 24 hrs. Synthetic mycolactone A/B with a final concentration of 3 ng/ml, 30 ng/ml, or 300ng/ml was added and further cultured for 12 and 24 hrs. Fixed cells were stained with fluorescent reagents. Red: cleaved caspase-3 (Rabbit anti-Cleaved Caspase-3 (1:1000)/Alexa Fluor 594 Goat Anti-Rabbit IgG); Blue: nuclear DNA (Hoechst 33342); and Green: intracellular actin (Alexa Fluor 488 Phalloidin). Cells were examined under a confocal laser scanning microscope (Olympus: FV10i-DOC Laser Scanning Microscope). As a positive control, actinomycin-D was added to the culture. Expression of cleaved caspase-3 was compared at 12 and 24 hrs after administration of mycolactone. Positive cell rate (number of cleaved caspase 3 positive cells/number of Hoechst 33342 positive cells, %) was calculated. (A and B) In the four conditions (12 and 24 hrs, 30 and 300 ng/ml mycolactone), the expression of cleaved caspase 3 was observed in the cytoplasm of SW10 Schwann cells (10–21%) and in some of L929 fibroblasts (2–3%). (C) Actinomycin-D showed the expression of cleaved caspase 3 to SW10 and L929 cells.</p

First Synthesis of EuS Nanoparticle Thin Film with a Wide Energy Gap and Giant Magneto-Optical Efficiency on a Glass Electrode

Novel magneto-optical thin films composed of europium sulfide (EuS) nanoparticles on a glass electrode exhibit large magneto-optical efficiency and a wide energy gap. EuS nanoparticle thin films are prepared by the electrochemical reduction of a single-source precursor, a Eu­(III) dithiocarbamate complex (tetraphenylphosphonum tetrakis­(diethyldithiocarbamate) europium­(III)). The EuS nanoparticle thin films were prepared on indium–tin oxide (ITO)-coated glass electrodes and characterized by electrochemical analysis, scanning electron microscopy, energy-dispersive X-ray spectroscopy, transmission electron microscopy, laser scanning microscopy, and absorption spectroscopy. Faraday rotation spectra for estimation of the magneto-optical efficiency have clear positive and negative peaks, which are attributed to the 4f–5d transitions of the EuS thin films. The positive and negative peaks of the Faraday rotation spectrum are 525 and 680 nm, which are directly related to the energy gap of the EuS nanoparticle thin film (2.4 eV). That spectrum indicates that the EuS nanoparticle thin films are blue shifted in comparison with 7 nm diameter EuS nanoparticles (2.2 eV). The Verdet constant of the thin film was 11 mdeg/cm Oe at 525 nm, which is approximately 10 times larger than that of previously reported EuS nanoparticles

TUNEL assay in SW10 and L929 cells.

<p>L929 fibroblasts and SW10 Schwann cells were cultured on BD Falcon 2 well culture slides for 24 hrs (2.0x10⁴ cells/well) before synthetic mycolactone A/B was added. The cells were fixed with 4% paraformaldehyde-PBS and washed with PBS at 24 hrs and 48 hrs. The TUNEL assay and mild hematoxylin nuclear staining were performed. Total cell number and number of TUNEL-positive cells were counted using photomicrographs. At 24 hrs, 30 ng/ml of mycolactone induced less apoptosis (brown nuclear staining) in fibroblasts (A) than in Schwann cells (B). Three ng/ml of mycolactone did not show significant TUNEL reaction, while 300 ng/ml produced a strong TUNEL reaction in both fibroblasts and Schwann cells. In the quantitative analysis (C), fibroblasts in 3 ng/ml of mycolactone showed no apoptosis at 24 and 48 hrs, but 30 and 300 ng/ml of mycolactone induced apoptosis in a concentration-dependent and time-dependent manner. Schwann cells also showed no apoptosis at 24 and 48 hrs with 3 ng/ml of mycolactone; however, 30 and 300 ng/ml of mycolactone induced more apoptosis in Schwann cells (91% at 48 hrs, 300 ng/mg) than in fibroblasts (48% at 48 hrs, 300 ng/ml). Bar = 100 μm.</p

Oxidative Functionalization of Cinnamaldehyde Derivatives: Control of Chemoselectivity by Organophotocatalysis and Dual Organocatalysis

The catalytic and chemoselective oxidation of cinnamaldehyde derivatives having a CC bond and formyl group was studied by using two organocatalysts. The visible-light-induced catalysis using rhodamine 6G as an organophotocatalyst promoted the methoxyhydroxylation of the CC bond in a chemoselective manner. In contrast, the cooperation between rhodamine 6G and <i>N</i>-heterocyclic carbene (NHC) allowed the oxidative esterification of formyl group

Aqueous-Medium Carbon–Carbon Bond-Forming Radical Reactions Catalyzed by Excited Rhodamine B as a Metal-Free Organic Dye under Visible Light Irradiation

The utility of rhodamine B as a water-soluble organic photocatalyst was studied in the cascade radical addition–cyclization–trapping reactions under visible light irradiation. In the presence of (<i>i</i>-Pr)₂NEt, the electron transfer from the excited rhodamine B to perfluoroalkyl iodides proceeded smoothly to promote the carbon–carbon bond-forming radical reactions in aqueous media. When <i>i</i>-C₃F₇I was employed as a radical precursor, the aqueous-medium radical reactions proceeded even in the absence of (<i>i</i>-Pr)₂NEt. In these reactions, the direct electron transfer from the excited singlet state of rhodamine B would take place. Furthermore, the cleavage of the C–I bond in less reactive <i>i</i>-PrI could be achieved by the reductive electron transfer from the excited rhodamine B, which was confirmed by the fluorescence quenching of rhodamine B with the addition of <i>i</i>-PrI

Effects of rbIFNT on IL-1β production and the role of IL-10.

<p>(A) THP-1 macrophages were incubated for 48 h with or without rbIFNT. After priming with LPS (100 ng/mL) for 3 h, cells were treated with nano-silica particles (100 µg/mL) for 6 h. Protein levels of pro-IL-1β and mature IL-1β in supernatants were detected by Western blot analyses. Representative photographs are shown. (B) Subsequently, total RNA was extracted and analyzed by real-time RT-PCR for expression of IL-1β mRNA. (C) IL-10 levels in supernatants were then determined using ELISA. (D) THP-1 macrophages were incubated with or without rbIFNT (27 IU/mL) in the presence of antibody against human IL-10 or control IgG, and then treated with LPS and nano-silica. IL-1β levels in supernatants were then determined using ELISA. Data are expressed as means ±SEM (n = 3); Significant differences were identified using ANOVA; ** <i>p</i><0.01.</p

Effects of rbIFNT on expression of scavenger receptors and the functional analysis of MARCO by siRNA knockdown.

<p>(A) THP-1 macrophages were incubated for 48 h with or without rbIFNT (27 IU/mL) and total RNA was then extracted and analyzed by real-time RT-PCR for MARCO, CD36, SR-BI, MSR1 and OLR1 mRNA. (B) THP-1 macrophages were transfected with MARCO siRNA (siMARCO) or control siRNA (siN.C.) and analyzed MARCO mRNA expression by real-time RT-PCR (C). THP-1 macrophages transfected with MARCO siRNA or control siRNA were incubated with green nano-silica particles (30 µg/mL) for 6 h. After washing cells, uptake of green nano-silica particles was analyzed the amount of nano-silica (mean fluorescence intensity; MFI) by flow cytometry. After nano-silica treatment, IL-1β levels in supernatants were determined using ELISA. Data are expressed as means ±SEM (n = 3–4); Significant differences were identified using t-test; ** <i>p</i><0.01 vs. control.</p

Effects of rbIFNT on nano-silica uptakes and actin polymerization.

<p>(A) THP-1 macrophages were treated with Green nano-silica for 6 h at the indicated concentrations. (B) THP-1 macrophages were incubated for 48 h with or without rbIFNT (2.7 and 27 IU/mL), and were then treated with Green nano-silica particles (30 µg/mL) for 6 h. After washing cells, uptake of Green nano-silica particles was analyzed by flow cytometry. (C) THP-1 macrophages were incubated for 1 h with or without cytochalasin D and were treated with Green nano-silica particles (30 µg/mL) for 6 h. After washing cells, uptake of Green nano-silica particles was analyzed by flow cytometry. Representative flow cytometry plots are presented. (D) THP-1 macrophages were incubated for 48 h with or without rbIFNT (27 IU/mL), and were then treated with nano-silica particles (30 µg/mL) for 6 h. Cells were stained with fluorescent-conjugated phalloidin and analyzed by flow cytometry. Nuclei were co-stained with Hoechst 33342. Representative confocal microscopic images of the F-actin assembly are presented. Quantitative analyses were performed and data are expressed as means ±SEM (n = 3); Significant differences were identified using ANOVA; *<i>p</i><0.05 and ** <i>p</i><0.01.</p